Methods for the efficient, non-destructive, and complete sorting of cells have widespread applications in basic biological and medical research. For example, cell sorting is commonly used in immunology, where cells displaying specific markers are segregated from other cells via an optical property such as fluorescence. Another application is in medical therapeutics, where a certain autologous or heterologous cell type is commonly desired for transplantation, as in therapy for neoplasia. Advances in the microfabrication of biocompatible materials, and of bioengineering in general, suggest that more effective cell sorting methods will also find use in tissue engineering applications.
Early cell sorting devices distinguished among cells based upon physical parameters. Such cell sorting techniques included filtration, which selects based on cell size, and centrifugation, which selects based on cell density. These methods are effective if the cell population of interest differs significantly in size or density from the other cells in a cell mixture. When the individual cell populations in the cell mixture are similar to each other in size and density, however, neither filtration nor centrifugation techniques can separate them effectively.
To overcome these disadvantages, techniques have been developed to distinguish and separate cell populations based on the display of surface markers or epitopes. These techniques differentiate between cell populations based on tagging elements attached to the cell surface, and have become significant cell-sorting tools. Fluorescence-activated cell sorting (FACS) employs a fluorescent antibody label or tag that binds a specific cell surface marker. Most such sorters operate primarily in a binary manner: selection is based solely upon whether or not a cell bears sufficient fluorescent labels to trigger separation based upon a preselected threshold fluorescence value. Because FACS sorters examine a single cell at a time, the rate of cell separation is relatively slow. Generally, a FACS sorter can provide a cell sorting rate of 103 cells/second. Higher cell sorting rates are possible, but they may damage some cells. A limited number of FACS sorters are present in laboratories because they are costly and must be operated by skilled technicians.
Another separation method that utilizes cell tagging is known as high gradient magnetic separation (HGMS). Magnetic based sorting was first employed in the mining industry, and relies on differences in the intrinsic magnetic properties of the sorted materials for its operation (see U.S. Pat. No. 2,056,426 to Frantz). In HGMS, a heterogeneous cell population or cell mixture, which includes a magnetically tagged cell subpopulation, passes through an applied magnetic field, and the cells of the subpopulation labeled with magnetic tags are selectively attracted towards the magnetic source. The magnetically tagged subpopulation is collected by adherence to the magnetic source, or to a cell collector near the magnetic source. One shortcoming of HGMS, which can be faster than FACS, is that the cell subpopulation of interest can be damaged during the HGMS process due to massing of cells at the collector. HGMS is again primarily binary in nature, as separation is based solely on the presence or absence of magnetic tags.
Binary separation techniques based on a parameter such as magnetism or fluorescence have found considerable use in cell sorting. A need exists, however, for separating cells in a non-binary manner, based on the intensity of a specified parameter, such as the intensity of a detected magnetic or fluorescent signal.
Recently a system and method for sorting cells based on the quantity of magnetic tags bound to the cell has been described (U.S. Pat. No. 6,120,735 to Zborowski et al.), which uses a channel in which the tagged cells flow through a magnetic field. The method is capable of higher throughput, while maintaining comparable to higher cell viability, relative to traditional FACS or HGMS. A population of particles having different magnetic susceptibilities is subjected to a magnetic field during flow to create a gradient in the flow stream. Divided flow compartments within the channel are used to generate fractionated efferent flow streams. The particles in these fractionated cell flow streams are not, however, strictly sorted, but rather are enriched in particular fractions. Thus the higher throughput of fractionated enrichment methods, while maintaining cell viability, is obtained by a sacrifice in purity. A need therefore exists for methods of cell sorting that allow high throughput, with the flexibility to perform non-binary separations, without sacrificing purity.
Another recently described method for sorting cells provides high throughput and avoids mere enrichment, but sacrifices cells by destroying all detected unwanted cells with a laser (U.S. Pat. No. 5,158,889 to Hirako et al., 1992).
Some cell sorting methods include the ability to separate a single file, continuous procession of fluid-suspended cells in a channel into a procession of individual droplets containing single cells, as described in U.S. Pat. No. 3,710,933 to Fulwyler et al. and U.S. Pat. Nos. 3,380,584 and 4,148,718 to Fulwyler. The procession of individual droplets is created by vibrating a flow chamber or orifice through which the flow passes, usually at a frequency on the order of 40,000 Hz. These droplets, each containing a single cell, may be ejected from an orifice. In this method, the single-file cells are separated from each other by a significant distance, resulting in a smaller number of cells passing a detection or ejection point per unit of time relative to the number in a method that employs a continuous progression of single-file, nearly adjacent cells. Thus, sorting throughput and efficiency are relatively low, because selected cells cannot be ejected from the procession as rapidly as in the case where the fluid is continuous. Also, there is much inflexibility and inefficiency associated with the manipulating of individual cells in a channel containing many cells. The speed of manipulating individual cells in a channel is inherently limited, for example, because the flow may need to be slowed or stopped to prevent cellular collisions in a channel or system of interconnected channels.
An example of such a fluid-suspended cell sorter is the jet-in-air sorter, which is commonly optimized for commercial mammalian cell sorting. Lymphoid cells having diameters ranging from 8 to 14 μm, and spermatocytes having a long dimension of up to 200 μm, are commonly sorted by such a device. Jet-in-air systems that are piezo-based must be tuned to the specific diameters of the cells to be sorted, so that it is difficult to sort subpopulations of cells having substantially different mean sizes. Tuning such a system to accommodate different cell sizes or fluid viscosities involves adjusting parameters such as flow tip diameter, sheath pressure, flow rate, droplet drive frequency, drive amplitude, droplet spacing, and droplet breakoff point.
Piezo-based systems also tend to be inefficient for other reasons, including their need to space out cells in the flow stream to prevent cell bunching, which reduces the capacity to quickly locate cells for sorting operations. For example, to avoid cell bunching, one fluid droplet out of ten may contain a cell. Consequently, for a flow rate of 32,000 drops per second, only 3,200 cells per second would be counted, a 10-fold lower efficiency compared to the use of a system wherein each droplet contains a cell.
A need therefore exists for a method and system capable of sorting a large range of particle sizes that do not require the flow tip to be changed or other fluidic parameters to be adjusted. Indeed, a need exists for cell sorting methods and systems that do not use flow tips, to eliminate the potential for clogging. A need also exists for a sorting system and method that can readily discriminate between clumps of cells and single cells without clogging, permitting clumps to be identified and sorted separately. There is an additional need for a sorting system and method that can be adjusted to accommodate solutions of varying viscosities by merely changing the frequency and power settings on the energy transducer. A further need exists for a cell sorting system that can economically attain throughput and efficiency levels superior to those of current systems by, for example, the use of massively parallel, multi-channel sorting. Fundamental needs still exist for improved methods to differentiate cells according to multiple parameters, and to separate cells into two or more groups based on the degree of a single parameter (non-binary decision making), without sacrificing separation purity or cell viability.
For research purposes, numerous improvements are desired for cell sorting systems. Overall, there is a need for greater efficiency so that, for example, the total time is shortened between obtaining a cell mixture (for example a blood sample) and using the separated cells experimentally. Furthermore, experiments commonly require the plating of small numbers of a specific cell type onto individual plates, dishes, wells, or arrays thereof. Because all known cell sorting methods first collect all cells of a given subpopulation into one place, rather than permitting removal of individual selected cells directly into well plate wells or other containers, more steps are required between collecting a sample and having the collected cells ready for experimentation. Considerable laboratory time and effort can be saved by direct delivery of a precisely known small number of individually selected cells into containers for use in experiments, rather than collecting the entire separated subpopulation into a single container and then subdividing the cells into experimental vessels. Thus a need exists for employing a means for the non-binary selective removal of viable cells from a mixture of cells directly into an experimental vessel. This need can be met through the use of acoustic ejection.
There is no method or system currently known for sorting cells in which individual viable cells are ejected from a fluid. Thus a need exists for a method and corresponding system for sorting cells by ejecting viable single cells from a fluid, which preferably use non-binary selection and can deliver precise numbers of cells from a fluid directly into experimental containers. A method for ejecting single cells from a fluid is generally disclosed in copending U.S. patent applications Ser. Nos. 09/727,391 and 09/999,166, filed on Nov. 29, 2000 and Nov. 29, 2001, respectively, for “Focused Acoustic Energy for Ejecting Cells from a Fluid”, inventors Mutz and Ellson, assigned to Picoliter Inc. (now Labcyte Inc. of Sunnyvale, Calif.). A method and system for cell sorting utilizing the acoustic ejection of individual selected cells contained in droplets offer increased flexibility and overall efficiency without reducing viability, as compared to existing methods, by virtue of their ability to deliver sorted cells directly into experimental containers and to sort cells into several, rather than just two, groups based on a single intrinsic or tagged property.